This work was supported by an Innovation and Technology Development award from the Center for the Development of Therapeutics at the Broad Institute of MIT and Harvard. The authors wish to thank the members of the senior leadership team and the review committee for their support of this work.

All the proteins were overexpressed in E.coli with good yield and purity (&#62;80%) following the literature protocols18. Biotinylation was carried out using a BirA-catalyzed reaction18. All small molecules were prepared at 1 mM stock solutions in 100% DMSO. The procedures described herein do not require specialized laboratory safety equipment or precautions. Standard laboratory personal protective equipment (PPE) should be used (i.e., lab coat, safety goggles, and gloves).
Proteins applied in this study are listed below: 
VHL: biotinylated VHL(53-213)/ElonginB (1-104)/ElonginC(17-112) complex with Avi-tag at the C-terminus of ElonginB. 
Brd4BD2: Non-tagged Brd4BD2(333-460) 
CRBN: biotinylated CRBN(319-442) with Avi-tag at the N terminus 
PPM1D: non-tagged or double His8-tagged PPM1D(1-420) at the N terminus
Small molecules applied in this study are listed below: 
MZ1 (MW = 1002.6 Da): PROTAC that binds to VHL and Brd4BD2 
BRD-2512 (MW = 841.4 Da): CRBN KD ~3 &#181;M, doesn&#39;t bind to PPM1D 
BRD-5110 (MW = 872.0 Da): CRBN KD ~3 &#181;M, PPM1D KD = 1-2 nM 
BRD-4761 (MW = 476.6 Da): doesn&#39;t bind to CRBN, PPM1D KD = 1-2 nM
1. Method 1: ITC (isothermal titration calorimetry)
NOTE: Titrations are performed using a micro-calorimeter with auto-injection.
<ol>
	<li>Buffer preparation: Prepare 3 L of buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 7.5.</li>
	<li>Dialysis: Dialyze VHL and Brd4BD2 (~ 500 &#181;L at 150 &#181;M each) against 1 L of the buffer prepared in step 1.1, 3 times at 4 &#730;C, for 4 h, 2 h, and approximately&#160;16 h, respectively. Save the buffer after the last dialysis for use in subsequent steps.</li>
	<li>Prepare samples on a 96-well plate with a plastic cover.
	<ol>
		<li>For each titration, prepare 400 &#181;L of solution for the cell, 125 &#181;L for the syringe, and 400 &#181;L of buffer for cleaning. Add samples to three consecutive wells on the plate. Since compound stock is prepared at 1 mM in 100% DMSO, add the same percentage of DMSO to the protein solutions to ensure the matching buffer in the cell and syringe. Add 2% DMSO to the final solutions.
		<ol>
			<li>Sample for the titration of VHL into MZ1: Prepare cell solution containing 392 &#181;L of buffer, 4 &#181;L of MZ1 at 1 mM (10 &#181;M final concentration), and 4 &#181;L of 100% DMSO. Prepare a syringe solution containing 122.5 &#181;L of VHL at 85.7 &#181;M, 2.5 &#181;L of 100% DMSO (84 &#181;M final concentration).</li>
			<li>Samples for the titration of VHL into the buffer (data will be used for background subtraction of data generated from sample 1.3.1.1): Prepare cell solution containing 392 &#181;L buffer, 8 &#181;L 100% DMSO. Prepare a syringe solution containing 122.5 &#181;L of VHL at 85.7 &#181;M, (84 &#181;M final concentration) and add 2.5 &#181;L of 100% DMSO.</li>
			<li>Samples for the titration of VHL into MZ1 and Brd4BD2: Prepare cell solution containing 392 &#181;L of 17.1 &#181;M Brd4BD2 (16.8 &#181;M final concentration), 3.36 &#181;L of MZ1 at 1 mM (8.4 &#181;M final concentration), and 4.64 &#181;L of 100% DMSO. Prepare a syringe solution containing 122.5 &#181;L of VHL at 85.7 &#181;M (84 &#181;M final concentration) and 2.5 &#181;L of 100% DMSO.</li>
			<li>Samples for the titration of VHL into Brd4BD2 (background of 1.3.1.3): Prepare cell solution containing 392 &#181;L of 17.1 &#181;M Brd4BD2 (16.8 &#181;M final concentration) and 8 &#181;L of 100% DMSO. Prepare a syringe solution containing 122.5 &#181;L of VHL at 85.7 &#181;M (84 &#181;M final concentration) and 2.5 &#181;L of 100% DMSO.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Run all four titrations on the micro-calorimeter. Each consists of 19 injections of 2 &#181;L syringe solution at a rate of 2 &#181;L/s at 120 s time intervals. Make an initial injection of protein (0.4 &#181;L) and discard it during data analysis. Perform all experiments at 25 &#730;C, while stirring at 600 rpm.</li>
	<li>Data analysis: Fit the data to a single-binding-site model to obtain the stoichiometry (n), the dissociation constant (KD), and the enthalpy of binding (&#916;H) using the analysis software provided by the manufacturer (Figure 2).</li>
</ol>
2. Method 2: BLI (biolayer interferometry)
<ol>
	<li>Perform BLI experiments using streptavidin (SA) coated sensors at room temperature (RT) with an acquisition rate of 5 Hz. During the automated BLI experiment, ensure the sensors are stationary within a single column&#160;and move between different columns of a 96-well, flat bottom, black plate with a maximum well volume of 392 &#181;L. Fill each well of the plate used in the experiment with 200 &#181;L of solution. 
	NOTE: BLI is only useful to detect protein-protein interactions (i.e., ternary complex formation). It is not sensitive enough to detect interactions between proteins and small molecules.
	<ol>
		<li>Buffer preparation: Prepare 100 mL of buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 0.05% P20, pH 7.5.</li>
		<li>Optimization for the immobilization step: Load the test sensor to 1-3 nm, as recommended by the manufacturer. A loading of ~1.0 nm is used for the procedures described here. To achieve this, dip the sensor into a solution containing VHL at 1.5 &#181;g/mL for 80 s.</li>
		<li>Carry out BLI kinetic measurements by applying seven SA sensors using the following sequence:
		<ol>
			<li>For the first baseline phase, dip into buffer for 60 s.</li>
			<li>For the immobilization phase, dip into a solution of VHL at 1.5 &#181;g/mL for 80 s.</li>
			<li>For the second baseline phase, dip into&#160;buffer for 60 s.</li>
			<li>For the association phase, dip into solution of a fixed concentration of Brd4BD2 at 2 &#181;M, fixed concentration of DMSO at 2%, and varying concentrations of MZ1 at 100 nM, 50 nM, 25 nM, 12.5 nM, 6.3 nM, 3.1 nM, and 0 nM (reference sensor) for 300 s.</li>
			<li>For the dissociation phase, dip into&#160;buffer for 600 s.</li>
		</ol>
		</li>
		<li>Perform data analysis using the manufacturer&#39;s software. kon, koff, and KD are reported for data fitting (Figure 3).</li>
	</ol>
	</li>
</ol>
3. Method 3: SPR (surface plasmon resonance)
NOTE: All SPR experiments are carried out using streptavidin (SA) coated sensor chips at RT. Although the NTA chip is used for the detection between protein and small molecules, it is to be used with caution when applied to the ternary complex, as a much higher background than the SA chip is observed, possibly due to electrostatic interactions between the charged chip surface and protein in the analyte.
<ol>
	<li>SPR for VHL-MZ1 interaction
	<ol>
		<li>Buffer preparation.
		<ol>
			<li>Prepare a 1 L buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 0.005% P20, pH7.5, and pass it through a 0.2 &#181;m filter unit.</li>
			<li>Remove 20 mL of buffer for future use (DMSO-free buffer) and refill with 20 mL of DMSO (2% DMSO in the final running buffer). Keep DMSO at 2% in all the samples described below (steps 3.1 and 3.2)</li>
		</ol>
		</li>
		<li>Activate the SA chip following the manufacturer&#39;s protocol and immobilize VHL to ~2000 RU (inject protein solution of 5 &#181;g/mL at 5 &#181;L/min).</li>
		<li>Prepare MZ1 in a 3-fold, 8-point serial dilution with the top concentration at 10 &#181;M on a 384-well, conical bottom, translucent, polypropylene plate with a maximum volume of 130 &#181;L. Cover the plate with self-adhesive, transparent plastic foils compatible with polypropylene microplates.
		<ol>
			<li>Prepare the top concentration with the DMSO-free buffer to ensure the 2% DMSO at the final concentration. To 147 &#181;L of DMSO-free buffer, add 1.5 &#181;L of MZ1 stock at 1 mM and 1.5 &#181;L of 100% DMSO.</li>
			<li>Prepare the 3-fold serial dilution with the running buffer containing 2% DMSO. To 100 &#181;L of the running buffer, add 50 &#181;L of solution at the top concentration prepared in step 3.1.3.1, and mix well to prepare the 2nd highest concentration.</li>
			<li>Then transfer 50 &#181;L of the solution prepared in step 3.1.3.2 to the next 100 &#181;L of running buffer, mix well to prepare the 3rd highest concentration, and so on.</li>
		</ol>
		</li>
		<li>Run SPR using the multi-cycle setup: Mode: High-performance; contact time: 120 s; dissociation time: 300 s; flow rate: 50 &#181;L/min.</li>
		<li>Perform data analysis using the evaluation software provided by the instrument manufacturer. Steady-state analysis indicated KD = 26 (&#177; 3) nM and Rmax of ~91% (&#177; 5%) binding achieved (Figure 4A)</li>
	</ol>
	</li>
	<li>SPR for VHL: MZ1: Brd4BD2 ternary complex
	<ol>
		<li>Prepare the buffer as described in step 3.1.1.</li>
		<li>Activate the SA chip following the manufacturer&#39;s protocol and immobilize VHL to ~100 RU. Inject a protein solution of 0.5 &#181;g/mL VHL at a flow rate of 5 &#181;L/min and contact time between 1-5 min until a surface density of ~100 RU is reached.</li>
		<li>Prepare samples for two 5-fold, 5-point single-cycle injections in a 96-well,clear, polystyrene, round bottom plate with a maximum volume of 323 &#181;L and cover it with self-adhesive, transparent, plastic foils that are compatible with polystyrene microplates as described in steps 3.2.3.1-3.2.3.2.
		<ol>
			<li>Negative control: analyte contains Brd4BD2 only.
			<ol>
				<li>Prepare top concentration at 25 &#181;M in running buffer with a volume of 200 &#181;L (#A5)</li>
				<li>Add 160 &#181;L each of Brd4BD2 at 2 &#181;M in the running buffer to the next 4 wells on the left (#A1-A4)</li>
				<li>Transfer 40 &#181;L of A5 to A4. Mix well.</li>
				<li>Transfer 40 &#181;L of A4 to A3. Mix well. Keep doing so until A1.</li>
			</ol>
			</li>
			<li>Ternary complex formation: analyte contains MZ1 and Brd4BD2.
			<ol>
				<li>Prepare the top concentration by adding 4 &#181;L of MZ1 at 20 &#181;M in 100%-DMSO solution to 196 &#181;L containing 25.5 &#181;M Brd4BD2 in DMSO-free buffer (#B5). The final concentrations are 25 &#181;M Brd4BD2, 100 nM MZ1, and 2% DMSO.</li>
				<li>Add 160 &#181;L each of Brd4BD2 at 2 &#181;M in the running buffer to the next 4 wells on the left (#B1-B4)</li>
				<li>Transfer 40 &#181;L of B5 to B4. Mix well.</li>
				<li>Transfer 40 &#181;L of B4 to B3. Mix well. Keep doing so until B1.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Run SPR using the single-cycle setup: Contact time: 100 s; dissociation time: 720 s; flow rate: 50 &#181;L/min. Apply three injections of buffer before each sample to ensure a stable background.</li>
		<li>Data analysis: Apply the third injection of the buffer as the background for subtraction. As the negative control, when MZ1 is not present, the SPR response between VHL and Brd4BD2 is negligible. When MZ1 is present, kinetic fitting indicates the interaction between VHL and MZ1-Brd4BD2 complex has kon = 7.9 (&#177; 1.5) *107 /M/s, koff = 0.014 s-1, and KD = 1 nM. (Figure 4B)</li>
	</ol>
	</li>
	<li>SPR for CRBN and small molecule interactions
	<ol>
		<li>Buffer preparation.
		<ol>
			<li>Prepare a 1 L buffer containing 50 mM Tris, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.005% P20, pH 7.5, and pass it through a 0.2 &#181;m filter unit.</li>
			<li>Remove 20 mL of buffer and refill 20 mL of 100% DMSO (2% DMSO in the final running buffer). It is important to keep DMSO at 2% in all the samples described below (steps 3.3, 3.4, and 3.5).</li>
		</ol>
		</li>
		<li>Activate the SA chip following the manufacturer&#39;s protocol and immobilize CRBN to ~800 RU.</li>
		<li>Prepare compounds in 3-fold, 6-pt serial dilution on a 384-well plate with a top concentration of 30 &#181;M as described in 3.1.3.</li>
		<li>Run SPR using the multi-cycle setup: Mode: High-performance,&#160;contact time: 60 s, dissociation time: 90 s; flow rate: 50 &#181;L/min. For this system, three buffer injections are sufficient to obtain a stable background. For other assay systems, it may be necessary to perform additional buffer injections.</li>
		<li>Perform data analysis using the manufacturer&#39;s software. 
		NOTE: Steady-state analysis indicates KD of BRD-2512 and BRD-5110 are both around 3 &#181;M. However, binding only reached ~ 70% Rmax binding at the top concentration. Both weak affinity and the shape of the sensorgram indicate that compound insolubility at high concentrations is likely to happen. Thus, the actual KD might be higher than 3 &#181;M. BRD-4761, which does not bind to CRBN, was included as a negative control.</li>
	</ol>
	</li>
	<li>SPR for PPM1D and small molecule interactions
	<ol>
		<li>Prepare buffer as described in step 3.3.1. Use a nitriloacetic acid (NTA) sensor chip.</li>
		<li>Apply single-cycle because the PPM1D warhead has a slow kon and koff. Regenerate the NTA chip using the manufacturer&#39;s default setup and immobilize PPM1D to ~ 1000 RU after each compound injection (inject protein solution of 5 &#181;g/mL at 5 &#181;L/min).</li>
		<li>Prepare compounds in 3-fold, 5-point serial dilution with the top concentration at 400 nM as described in 3.1.3.</li>
		<li>Run SPR using the single-cycle setup: Mode: high-performance; contact time: 90 s; dissociation time: 600 s; flow rate: 50 &#181;L/min. Apply three injections of buffer before each compound to ensure a stable background.</li>
		<li>Data analysis. Apply the third injection of the buffer as the background for subtraction. As the negative control, BRD-2512 does not show binding to PPM1D. For BRD-4761 and BRD-5110, kinetic fitting indicated the KD for both to be 1-2 nM.</li>
	</ol>
	</li>
	<li>SPR for CRBN:PROTAC: PPM1D ternary complex
	<ol>
		<li>Prepare buffer as described in step 3.3.1.</li>
		<li>Activate the SA chip following the manufacturer&#39;s protocol and immobilize CRBN to ~35 RU (inject a protein solution of 0.5 &#181;g/mL at 5 &#181;L/min).</li>
		<li>Prepare three compounds in 3-fold, 6-point serial dilution with a top concentration of 30 &#181;M while keeping [PPM1D] at 1 &#181;M in all samples, including blanks, using the method described in step 3.2.3.</li>
		<li>Run SPR using the multi-cycle setup: Mode: High-performance; contact time: 60 s; dissociation time: 90 s; flow rate: 50 &#181;L/min.</li>
		<li>Perform data analysis using the manufacturer&#39;s software. 
		NOTE: While BRD-2512 and BRD-4761 show no/negligible response, BRD-5110 clearly shows the &#34;hook effect&#34; at the steady state with fast on/off kinetics (Figure 5A-C). The experimental binding response from BRD-5110 (Figure 5C) is in between the predicted response from the simulation when assuming KD (CRBN, cpd) is 3 or 10 &#181;M (Figure 5E), suggesting the ternary KD and binary KD are very similar. There is no apparent cooperativity of PPM1D: BRD-5110: CRBN complex formation.</li>
	</ol>
	</li>
</ol>

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

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The authors have no competing financial interests or other conflicts of interest.

Biophysical characterization of the binary and ternary interactions between PROTAC molecules and their protein binding partners can provide unique and complementary insights relative to widely used cellular systems. Understanding the affinity between each warhead of a PROTAC molecule and its protein binding partners can help guide medicinal chemistry efforts toward the optimization of those interactions. Previously published crystal structures of ternary PROTAC complexes have revealed that atoms in the linker region can ...

The polyubiquitination of proteins in the cell is a tightly regulated process that involves enzymes in the Ubiquitin Ligase family1,2. The terminal enzymes in the pathway are the E3 ubiquitin ligases that covalently attach ubiquitin molecules to their protein-binding partners3. The polyubiquitination of those protein binding partners targets them for proteolytic degradation by the proteasome4. This system is part of the protein homeostasis process that has been therapeutically leveraged to induce the degradation of proteins involved in disease5. Small molecules that induce the interaction between E3 ubiquitin ligases, such as Von Hippel-Lindau E3 ligase (VHL) or cereblon (CRBN), are typically composed of an E3 ligase binding warhead connected by a flexible linker to a warhead that binds to the protein being targeted for degradation. These heterobifunctional molecules are commonly referred to as proteolysis targeting chimeras or PROTACS6.
The development of PROTACS involves evaluating the ability of molecules to induce the degradation of proteins in cells. Many cellular assay systems have been developed that monitor the induced interaction between the target protein and E3 ligase components, such as VHL or CRBN, upon treatment of the cells with a PROTAC molecule. One such cellular assay, the nanoluc-Halotag system7, involves an E3 ligase fused to the Halotag acceptor and a target protein tagged with a nanoluc donor. Ternary complex formation brings the nanoluc donor and Halotag acceptor into proximity allowing the transfer of energy from the donor to the acceptor resulting in the emission of light. Variations of this system can be used to assess the cellular permeability of PROTACS molecules8 or changes in the relative level of target protein ubiquitination9. While these cellular systems are essential for driving the optimization of PROTACS, the formation of complexes between E3 ligases and proteins targeted for degradation is a multi-step process10,11. The kinetics and thermodynamics of the binary and ternary interactions involved contribute to efficiency ubiquitination and resulting degradation of the protein12,13,14.
Herein are described protocols that can be adapted for the biophysical characterization of ternary complex formation induced by PROTACS using surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC). SPR and ITC protocols for the MZ1 PROTAC molecule that induces ternary complex formation between Brd4BD2 and VHL derived from literature reports13,15 and described here were able to recapitulate the reported results with some modification of the reported procedures, which will be discussed. A description of a BLI assay used to evaluate ternary complex formation between MZI, Brd4BD2, and VHL is included in this report. Affinity measurements from BLI were consistent with those observed in SPR and ITC. A previously published protocol in which an SPR assay was developed for assessing the PROTAC-induced ternary complex formation between PPM1D, a Ser/Thr protein phosphatase whose expression is induced in a p53-dependent manner16, and CRBN is also described. In this instance, the PROTAC molecule has a nanomolar affinity for PPM1D but only a micromolar affinity for CRBN. In this case, the binding of the PROTAC molecule to CRBN is not saturable, resulting in the commonly observed &#34;hook effect&#34;. The hook effect is a property of three body systems in which there are two species that can form a heterotrimeric complex when both are bound to a bridging molecule (Figure 1)17. The hook effect is observed when the bridging species is in excess concentration relative to the two other species. The resulting state is one in which the binary interactions outcompete the ternary interactions. The systems where the hook effect is observed require specific experimental design considerations discussed in this report. General concepts and reagent requirements for evaluating the utilization of biophysical assays for the evaluation of PROTAC-induced ternary complex formation are provided.

<table><tbody><tr><td>96-plate</td><td>Greiner</td><td>655076</td><td>flat-bottom, black plates used In BLI experiments</td></tr><tr><td>96-well plate</td><td>Nunc</td><td>73520-120</td><td>Plate use for ITC sample preparation</td></tr><tr><td>96-well plate</td><td>Greiner</td><td>650101</td><td>Plate used to prepare samples for SPR experiments</td></tr><tr><td>Auto iTC200 micro-calorimeter</td><td>Malvern Panalytical</td><td></td><td>Instrument used to perform ITC experiments. Product discontinued.</td></tr><tr><td>Biacore S200</td><td>Cytiva</td><td>29136649</td><td>Instrument used to perform SPR experiments</td></tr><tr><td>MZ1</td><td>ProbeChem</td><td>PC-60099</td><td>PROTAC that binds to VHL and Brd4BD2</td></tr><tr><td>NTA sensor chip</td><td>Cytiva</td><td>BR100532</td><td>SPR chip used to perform SPR experiments involving PPM1D</td></tr><tr><td>Octet Red-384</td><td>Sartorius</td><td></td><td>Instrument used to perform BLI experiments. Product discontinued.</td></tr><tr><td>Plate cover</td><td>Malvern</td><td>PQA0001</td><td>Cover for Nunc 96-well plate (73520-120)</td></tr><tr><td>Plate cover</td><td>Cytiva</td><td>28975816</td><td>Plate cover for Greiner plate (650101)</td></tr><tr><td>Series S SA sensor chip</td><td>Cytiva</td><td>BR100531</td><td>SPR chip used to perform SPR experiments involving MZ1:VHL:BRD4</td></tr><tr><td>Streptavidin (SA) Dip and Read Biosensors</td><td>Sartorius</td><td>18-509</td><td>Coated sensors used in BLI experiments</td></tr></tbody></table>

the development and application of biophysical assays for evaluating ternary complex formation induced by proteolysis targeting chimeras (protacs)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Characterization of VHL: MZ1 binary complex and VHL: MZ1: Brd4BD2 ternary complex can be found in Figure 2 (ITC), Figure 3 (BLI), and Figure 4 (SPR) using a very similar buffer. The KD extracted from orthogonal assays is consistent. The cooperativity can be calculated by KD (binary) / KD (ternary), which is highly positive (15 from ITC or 26 from SPR).
Character...

Watch this Scientific Journal Video about Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes at JoVE.com

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

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Biochemistry

3.1K Views.  The Broad Institute of MIT and Harvard. Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC). 

Video: Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

Cancer Research

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN&#8216;s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple ...

Chemistry

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Immunology and Infection

Quantitative-Irreversible Tethering: Screening Electrophilic Ligands Efficiently

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

Compounds that form covalent bonds with specific target proteins offer a variety of advantages&#160;as chemical probes and therapeutic agents. Most commonly, mildly reactive, electrophilic small molecules are employed to form covalent bonds with select cysteine side chains in specific proteins. Electrophile-first approaches of ligand discovery, whereby a library of electrophilic small molecules are screened against a protein target, have become popular as they avoid the need for time-consuming downstream installation of an electrophilic warhead. Such screening is complicated, however, as electrophilic ligands can exhibit a wide range of different rates of spontaneous reaction with cysteines. Quantitative-irreversible tethering (qIT) offers a fluorescence-based method for hit identification and development that normalizes data for these differences in intrinsic compound reactivity. Rates of reaction of individual compounds with a target protein are determined and compared to compound reactivity with the unstructured tripeptide glutathione (this being a proxy for spontaneous compound reaction), enabling the identification of compounds that preferentially react with the protein of interest. This methodology has been successfully applied to identify selective covalent fragments against several drug targets, including SARS-CoV-2 main protease, cyclin-dependent kinase 2, and RAP27A. Here, we demonstrate the application of qIT to a target protein to generate a quantitative and robust data set, allowing prioritization of hit ligands for future development.

The quantitative irreversible tethering (qIT) assay is a fluorescence-based method of identifying covalent fragments that selectively engage with a target protein. Here, a detailed protocol is outlined to describe the qIT assay, with example results included.

Compounds that form covalent bonds with specific target proteins offer a variety of advantages&#160;as chemical probes and therapeutic agents. Most ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

Summary

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Chapters in this video

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells